Background and Objective: Pseudomonas aeruginosa has become a frequent cause of nosocomial infections, particularly in intensive care units (ICUs). Many reports have documented high rates of resistance in this species to commonly-used broad-spectrum antibiotics. The aim of this study was to assess the in vitro activity of some antibiotics against Pseudomonas aeruginosa strains to determine the susceptibility patterns of isolates to different antibiotics. Materials and Methods: A total of 233 Pseudomonas aeruginosa isolates obtained from various clinical specimens of hospitalized children in Ali-Asghar hospital of Tehran (Iran) were considered for susceptibility test. These strains were tested against 12 different antibiotics by a disk diffusion method. Of these isolates, 33.9% were from trachea, 31.8% from urine, 6.9% from eye, 5.2% from blood, 5.1% from ear, 1.3% from cerebrospinal fluid, 1.2% from stool, and 14.6% from other sites. In addition, 48.5% of P. aeruginosa strains were isolated from patients in ICUs. Results: The most active antimicrobials were amikacin and other active compounds were gentamicin, ceftazidime, and ciprofloxacin respectively. Isolates from ICUs were more resistant to amikacin and gentamicin as compared to those from non-ICU wards (p<0.05). Isolates from trachea were more resistant to amikacin, gentamicin, ciprofloxacin and ceftazidime than those from other sites (p<0.05). Conclusion: Our study showed that amikacin was the most active agent against P. aeruginosa followed by gentamycin, ceftazidime, and ciprofloxacin. According to our in vitro study results, active antibiotic susceptibility testing and surveillance should be continued in order to curtail the problem of antibiotic resistance.